甾体化合物ASC26对ABCB1介导的肿瘤细胞耐药性的逆转作用及其机制研究

发布时间：2018-07-12 21:09

本文选题：多药耐药 + ABCB1抑制剂　；参考：《郑州大学》2017年硕士论文

【摘要】：多药耐药性的产生,是导致肿瘤化疗失败的主要因素。研究报道ABC转运体在肿瘤耐药中发挥着重要作用,ABC转运体家族成员之一的ABCB1,是最早发现的ABC转运体,ABCB1的过表达是导致肿瘤耐药性产生的关键因素之一。寻找高效低毒的ABCB1选择性抑制剂已成为科研工作者的研究热点。本课题利用ABCB1过表达的肿瘤耐药细胞株,对一系列甾体衍生物进行了逆转耐药活性研究,旨在筛选出高效低毒的ABCB1选择性抑制剂,并研究其逆转耐药的作用机制,为解决肿瘤耐药性问题提供研究模型。具体研究内容如下:1.以ABCB1过表达的结肠癌耐阿霉素SW620/Ad300细胞株为基础,建立逆转剂筛选平台,采用MTT法对一系列甾体衍生物进行筛选,旨在寻找高效低毒的耐药逆转剂。结果表明,新型甾体化合物ASC26在浓度为5μM时能显著逆转ABCB1调控的耐药,并且对细胞的抑制率仅为7.39%。2.利用MTT法评价ASC26对七对肿瘤耐药及其亲本细胞的增殖抑制作用,结果发现ASC26在8μM时,对这七对肿瘤耐药及亲本细胞的抑制率均小于15%,即没有细胞毒性。3.进一步研究ASC26在浓度为8μM时的体外逆转耐药活性。结果表明,ASC26能使高表达ABCB1的肿瘤耐药细胞对紫杉醇(ABCB1的底物)的敏感性显著增加,而对顺铂(非ABCB1的底物)的抗肿瘤活性则无明显影响。此外,对于ABCG2过表达的耐药细胞,ASC26未表现出逆转耐药的作用。4.利用高效液相法检测ASC26对ABCB1高表达的肿瘤耐药细胞及其亲本细胞内紫杉醇蓄积和外排的影响。结果发现,ASC26能显著增加紫杉醇在ABCB1高表达的肿瘤耐药细胞中的蓄积,抑制ABCB1高表达的肿瘤耐药细胞对紫杉醇的外排,提高细胞内紫杉醇含量,而对亲本肿瘤细胞内紫杉醇的蓄积和外排无明显作用。5.流式细胞术检测ASC26对ABCB1高表达的SW620/Ad300细胞内罗丹明(Rho123)蓄积的影响。结果表明,ASC26能显著增加SW620/Ad300细胞内Rho123的滞留率,但对亲本SW620细胞内Rho123的蓄积无明显影响。6.免疫荧光检测ASC26对SW620/Ad300细胞内ABCB1转运蛋白表达位点的影响。ASC26作用于SW620/Ad300细胞72 h后,免疫荧光法检测ASC26对ABCB1表达位点的作用。结果表明,ASC26不改变细胞内ABCB1的蛋白表达位点。8.利用Western blot检测ASC26对ABCB1蛋白表达水平的影响。由Western blot结果可知,ASC26分别作用于SW620/Ad300耐药细胞0 h,24 h,48 h,72h后,并不改变细胞内ABCB1的蛋白表达水平。9.分子对接实验研究ASC26与人同源ABCB1分子模型的相互作用关系。分子对接实验表明,化合物ASC26能结合在ABCB1转运蛋白的跨膜结构域的药物结合口袋上,从而抑制ABCB1对化疗药物的外排转运。本课题发现了一个选择性ABCB1抑制剂,新型甾体化合物ASC26,在无毒浓度下,能显著逆转ABCB1介导的肿瘤耐药。进一步的机制研究表明,ASC26的逆转耐药机制是通过抑制ABCB1转运泵的外排转运功能,而不是通过影响ABCB1的蛋白表达来发挥逆转耐药作用。ASC26能与ABCB1转运蛋白的跨膜结构域的药物结合口袋结合,抑制ABCB1对抗肿瘤药物的外排作用,增加细胞内抗肿瘤药物的浓度,从而提高肿瘤细胞对化疗药物的敏感性,达到逆转肿瘤耐药的效果。这一研究成果将为解决肿瘤耐药性问题提供新的研究方案和研究模型。
[Abstract]:The production of multidrug resistance is the main factor leading to the failure of tumor chemotherapy. The study reports that the ABC transporter plays an important role in the drug resistance of the tumor. ABCB1, one of the members of the ABC transporter family, is the earliest found ABC transporter. The overexpression of ABCB1 is one of the key factors leading to the production of tumor resistance. The search for high efficiency and low toxicity ABCB1 Selective inhibitors have become the research hotspots of researchers. This topic uses ABCB1 overexpressed tumor resistant cell lines to reverse the drug resistance of a series of steroid derivatives. The purpose of this study is to screen out highly toxic and low toxic ABCB1 selective inhibitors, and to study the mechanism of its reversal of drug resistance, in order to solve the problem of drug resistance. The specific research contents are as follows: 1. based on the ABCB1 overexpressed adriamycin resistant SW620/Ad300 cell line of colon cancer, the reverse agent screening platform was established and a series of steroid derivatives were screened by the MTT method. The results showed that the new steroid compound ASC26 was at the concentration of 5 u M. The resistance to ABCB1 regulation was significantly reversed and the inhibitory rate to the cells was only 7.39%.2. using MTT to evaluate the inhibitory effect of ASC26 on the proliferation of tumor resistance and their parent cells by ASC26. The results showed that the inhibition rate of the seven against tumor resistance and parental cells was less than 15% when ASC26 was at 8 micron, that is, no cytotoxic.3. was further studied for ASC26. The reversal of drug resistance in vitro at a concentration of 8 M showed that ASC26 could significantly increase the sensitivity of the tumor resistant cells with high expression of ABCB1 to Taxol (ABCB1 substrate), while the antitumor activity of cisplatin (non ABCB1 substrate) was not significantly affected. In addition, ASC26 did not reverse the reversal of resistance to ABCG2 overexpressed drug-resistant cells. The effect of.4. on the effect of ASC26 on the accumulation and excretion of paclitaxel in ABCB1 high expression of tumor resistant cells and their parent cells by high performance liquid phase method. The results showed that ASC26 could significantly increase the accumulation of taxol in the tumor resistant cells with high expression of ABCB1, and inhibit the outer row of paclitaxel by the high expression of ABCB1. Intracellular paclitaxel content, but no significant effect on the accumulation and outer row of paclitaxel in the parent tumor cells.5. flow cytometry was used to detect the effect of ASC26 on the accumulation of Luo Danming (Rho123) in SW620/Ad300 cells with high expression of ABCB1. The results showed that ASC26 could significantly increase the retention rate of Rho123 in SW620/Ad300 cells, but Rho12 in the parent SW620 cell Rho12. The effect of the accumulation of 3 on the expression of ABCB1 transporter protein in SW620/Ad300 cells by.6. immunofluorescence was not significantly affected by.ASC26 on SW620/Ad300 cell 72 h, and the effect of ASC26 on the ABCB1 expression site was detected by immunofluorescence. The results showed that ASC26 did not change the ABCB1 protein expression loci in the cells. The effect of C26 on the expression level of ABCB1 protein. The result of Western blot shows that ASC26 acts on SW620/Ad300 resistant cells 0 h, 24 h, 48 h, and 72h, it does not change the ABCB1 protein expression level in the cell and the interaction relationship between the.9. molecular docking experiment and the human homologous molecular model. 6 can bind to the drug binding pocket of the transmembrane domain of the ABCB1 transporter, which inhibits the translocation of ABCB1 against chemotherapeutic agents. A selective ABCB1 inhibitor, a new steroid compound, ASC26, can be found to reverse the ABCB1 mediated swelling resistance of the tumor significantly in the non toxic concentration. Further mechanism studies show the inverse of ASC26 The mechanism of trans resistance is to inhibit the efflux transport of the ABCB1 transport pump, not by affecting the protein expression of ABCB1 to reverse the resistance of.ASC26 to the drug binding pocket of the trans membrane domain of the ABCB1 transporter, inhibit the use of ABCB1 against tumor drugs, and increase the concentration of intracellular antitumor drugs. To improve the sensitivity of tumor cells to chemotherapeutic drugs and to reverse the effect of tumor resistance, this research will provide a new research scheme and model for solving the problem of tumor resistance.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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